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AIM: Growth hormone (GH) directly interacts with the enterocyte stimulating ion absorption and reducing ion secretion induced by agonists of cAMP. Since nitric oxide (NO) is involved in the regulation of transepithelial ion transport and acts as a second messenger for GH hemodynamic effects, we tested the hypothesis that NO may be involved in the resulting effects of GH on intestinal ion transport. METHODS: Electrical parameters reflecting trans-epithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers and exposed to GH and cholera toxin (CT) alone or in combination, in the presence or absence of the NO synthase (NOS) inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME). Similar experiments were conducted to determine cAMP and nitrite/nitrate concentrations. NOS expression was assayed by Western blot analysis. RESULTS: L-NAME causes total abrogation of absorptive and anti-secretory effects by GH on intestinal ion transport. In addition, L-NAME was able to inhibit the GH-effects on intracellular cAMP concentration under basal conditions and in response to CT. GH induced a Ca(2+)-dependent increase of nitrites/nitrates production, indicating the involvement of the constitutive rather than the inducible NOS isoform, which was directly confirmed by Western blot analysis. CONCLUSION: These results suggest that the GH effects on intestinal ion transport, either under basal conditions or in the presence of cAMP-stimulated ion secretion, are mediated at an intracellular level by the activity of cNOS.

BACKGROUND & AIMS: Severe diarrhea and enteropathy of unknown origin are frequent in patients infected with human immunodeficiency type 1 virus (HIV-1). The HIV-1 transactivating factor protein (Tat) is a key factor in the pathogenesis of acquired immunodeficiency syndrome. We investigated whether Tat could directly induce ion secretion and cell damage in enterocytes. METHODS: Electrical parameters (ion transport studies) were measured in Caco-2 cell monolayers and in human colonic mucosa specimens mounted in Ussing chambers. The effect of Tat on intestinal mucosa integrity was determined by monitoring the transepithelial electrical resistance of Caco-2 cell monolayers. (3)H-thymidine incorporation and cell count were used to evaluate the effect of Tat on cell growth. Intracellular calcium concentrations were measured at the single-cell level using microfluorometry technique. RESULTS: Tat protein induced ion secretion in Caco-2 cells and in human colonic mucosa similar to that induced by bacterial enterotoxins. It also significantly prevented enterocyte proliferation. In both instances, the effect of Tat was maximum at concentrations within the range detected in the sera of HIV-1-infected patients. Anti-Tat antibodies inhibited both effects. Ion secretion and the antiproliferative effects were mediated by L-type Ca(2+) channels. An increase in intracellular calcium concentration in Caco-2 cells was found after addition of Tat. CONCLUSIONS: These results indicate that Tat may be involved in HIV-1-related intestinal disease through direct interaction with enterocytes.

BACKGROUND: Growth hormone (GH) stimulates intestinal growth and differentiation and promotes water and ion absorption in the rat intestine. Epidermal growth factor has similar effects, which involve tyrosine kinase activity. The effects of growth hormone on ion transport and cell growth and the role of tyrosine kinase in these effects were examined in a human-derived intestinal cell line (Caco-2). METHODS: For transport study, electrical parameters were measured in human intestinal Caco-2 cell monolayers mounted in Ussing chambers. Cell growth was monitored by counting and 3H-thymidine incorporation in the presence and absence of growth hormone. The role of tyrosine kinase was investigated by using its specific inhibitor genistein. RESULTS: The addition of growth hormone induced a rapid, Cl- -dependent, decrease in short-circuit current without affecting tissue conductance, which is consistent with an anion-absorptive effect. Incubation with growth hormone increased cell count by 85% and 3H-thymidine incorporation by 64% versus the count in control specimens. The absorptive and trophic effects of growth hormone were dose-dependent, and the maximum effective concentration was identical for each effect. Genistein blocked the growth hormone effect on ion transport and cell growth. CONCLUSIONS: Growth hormone stimulates ion absorption and cell growth in human enterocytes. Both effects result from a direct growth hormone-enterocyte interaction, and both require tyrosine kinase activity. Growth hormone may have therapeutic potential in intestinal diseases characterized by epithelial atrophy and loss of water and electrolytes.

Background & Aims: Severe diarrhea and enteropathy of unknown origin are frequent in patients infected with human immunodeficiency type 1 virus (HIV-1). The HIV-1 transactivating factor protein (Tat) is a key factor in the pathogenesis of acquired immunodeficiency syndrome. We investigated whether Tat could directly induce ion secretion and cell damage in enterocytes. Methods: Electrical parameters (ion transport studies) were measured in Caco-2 cell monolayers and in human colonic mucosa specimens mounted in Ussing chambers. The effect of Tat on intestinal mucosa integrity was determined by monitoring the transepithelial electrical resistance of Caco-2 cell monolayers. 3 H-thymidine incorporation and cell count were used to evaluate the effect of Tat on cell growth. Intracellular calcium concentrations were measured at the single-cell level using microfluorometry technique. Results: Tat protein induced ion secretion in Caco-2 cells and in human colonic mucosa similar to that induced by bacterial enterotoxins. It also significantly prevented enterocyte proliferation. In both instances, the effect of Tat was maximum at concentrations within the range detected in the sera of HIV-1–infected patients. Anti-Tat antibodies inhibited both effects. Ion secretion and the antiproliferative effects were mediated by L-type Ca 2+ channels. An increase in intracellular calcium concentration in Caco-2 cells was found after addition of Tat. Conclusions: These results indicate that Tat may be involved in HIV-1–related intestinal disease through direct interaction with enterocytes.

The role of nitric oxide (NO) in the intestinal basal ion transport and under conditions of enterotoxin-induced ion secretion is controversial. Namely it is not clear whether NO enhances or counteracts intestinal ion secretion and whether the effects on transport result from a direct interaction with the enterocyte. The cell origin of NO is also unclear. We have tested the hypothesis that NO produced by the enterocyte directly regulates ion transport processes either in basal condition or in response to cholera toxin-induced secretion. Electrical variables reflecting transepithelial ion transport were measured in Caco-2 cell monolayers mounted in Ussing chambers exposed to the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester, in the presence or absence of cholera toxin. cAMP concentrations were also measured. NO release was determined by nitrite-nitrate concentration. NO synthase activities were assayed by Western blot analysis. Nomega-nitro-l-arginine methyl ester had a secretory effect, as judged by increased basal short-circuit current and cAMP concentration. It also increased cholera toxin-induced electrical response and cAMP production. Either cholera toxin or the cAMP analog 8-bromo-cAMP induced a rapidly progressive and Ca2+-dependent increase in NO concentration, suggesting a homeostatic up-regulation of the constitutive form of NO synthase. Western blot analysis showed an increase in constitutive NO synthase enzyme isoform. These results indicate that the enterocyte regulates its own ion transport processes, either in basal condition or in the presence of active secretion, through the activation of a constitutive NO synthase-NO pathway, functioning as a braking force of cAMP-induced ion secretion.

Background: Growth hormone (GH) stimulates intestinal growth and differentiation and promotes water and ion absorption in the rat intestine. Epidermal growth factor has similar effects, which involve tyrosine kinase activity. The effects of growth hormone on ion transport and cell growth and the role of tyrosine kinase in these effects were examined in a human-derived intestinal cell line (Caco-2). Methods: For transport study, electrical parameters were measured in human intestinal Caco-2 cell monolayers mounted in Ussing chambers. Cell growth was monitored by counting and 3 H-thymidine incorporation in the presence and absence of growth hormone. The role of tyrosine kinase was investigated by using its specific inhibitor genistein. Results: The addition of growth hormone induced a rapid. Cl - -dependent, decrease in short-circuit current without affecting tissue conductance, which is consistent with an anion-absorptive effect. Incubation with growth hormone increased cell count by 85% and 3 H-thymidine incorporation by 64% versus the count in control specimens. The absorptive and trophic effects of growth hormone were dose-dependent, and the maximum effective concentration was identical for each effect. Genistein blocked the growth hormone effect on ion transport and cell growth. Conclusions: Growth hormone stimulates ion absorption and cell growth in human enterocytes. Both effects result from a direct growth hormone-enterocyte interaction, and both require tyrosine kinase activity. Growth hormone may have therapeutic potential in intestinal diseases characterized by epithelial atrophy and loss of water and electrolytes.